Understanding the molecular basis of anatomical variation is a fundamental challenge in biology. In some cases, the genes that control anatomical defects in humans instead underlie normal variation in other species; therefore, a comprehensive understanding of the general molecular mechanisms of diversity promises greater insights about human health. In certain breeds of domestic pigeon, genetic mapping and developmental studies show that regulatory changes in two genes that control limb-type identity (fore- or hindlimb) and outgrowth are associated with the replacement of scales by feathers on the feet. These changes in the skin are accompanied by defects in muscle and tendon patterning, thereby demonstrating that this phenotype is not limited to epidermal appendages. In humans, mutations in these same genes cause striking limb malformations, including clubfoot, Holt-Oram syndrome, and Liebenberg syndrome. This project seeks to understand how these genes function to specify tissue identity (e.g., scales or feathers) and regulate patterning of lim musculature. The pigeon is an ideal system in which to pursue these goals because it features tremendous morphological variation within a single species, thereby facilitating genome-wide association studies, traditional genetic mapping, and functional developmental biology. This project will pursue three separate aims. Aim 1 will identify cis-regulatory mutations in the two genes associated with transformations in limb-type identity. Enhancer mutations will be fine-mapped using a combination of whole-genome resequencing of breed with foot feathers (muffs), and massively parallel chromatin immunoprecipitation sequencing (ChIP-seq) to identify enhancer activity. The impact of candidate mutations will be tested using in ovo reporter assays. Aim 2 will determine the gene regulatory network governing skin appendage fate. This aim will employ embryonic tissue transplantation experiments and high-throughput RNA sequencing to determine how and when hindlimb skin identity is specified. Aim 3 will determine the functional impact of changes in limb-type gene expression on muscle and skin patterning. This aim will test the contributions of the two candidate genes to hindlimb skin and muscle mis- patterning in an F2 intercross. Additionally, experimental embryonic manipulations will determine whether specific regulation of these genes is necessary for normal hindlimb muscle pattern and epidermal appendage fate. Together, these complementary genetic, genomic, and developmental approaches will identify the molecular basis of astonishing variation in an innovative model system, thereby opening new avenues to understand the roles of specific genes in normal and disease variation among vertebrates in general.